N. Martínez-Lendech A. Córdoba-Aguilar (&) Martín A. Serrano-Meneses. Introduction

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1 J Ethol (2007) 25: DOI /s ARTICLE Body size and fat reserves as possible predictors of male territorial status and contest outcome in the butterfly Eumaeus toxea Godart (Lepidoptera: Lycaenidae) Norma Martínez-Lendech Æ Alex Córdoba-Aguilar Æ Martín A. Serrano-Meneses Received: 3 August 2006 / Accepted: 20 February 2007 / Published online: 20 March 2007 Ó Japan Ethological Society and Springer 2007 Abstract We used adults of the butterfly Eumaeus toxea for two purposes description of male territorial behavior and investigation of whether body size and muscle fat reserves correlated with social status (resident or intruder) and the probability of winning a territorial contest in dyadic encounters. Males perched in places near cycads (Zamia furfuracea), where females lay eggs. Resident males (those with faithfulness to the same site for several days) used two types of flight of different duration, short (ca. 5 s) and long (ca. 17 s), against male conspecifics. Because of this difference the latter were regarded as true contests. Males that copulated were residents and were also larger than males not observed in copulation. Residents and winners of contests were larger and fatter than intruders (males entering the site that faced a resident with a long-lasting flight) and losers of contests, respectively, possibly because these last two categories of male were individuals with already exhausted energy stores. Body size and fat reserves seemed to correlate with status for resident and intruder males but not for winner and loser males. This possibly means that body size reflects male energy condition. This is supported by the fact that large, resident males are confronted in short contests, in contrast with small, resident males. Our study suggests that the role of size and fat reserves during contests cannot be discounted in butterflies. Keywords Body size Eumaeus toxea Male contests Fat reserves N. Martínez-Lendech A. Córdoba-Aguilar (&) M. A. Serrano-Meneses Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, Apdo. Postal, , Ciudad Universitaria, Mexico, DF, Mexico acordoba@ecologia.unam.mx Introduction According to sexual selection theory, when males compete for access to females, individuals with more elaborate traits (e.g. songs, courtship, and size, among others; reviewed by Andersson 1994) will leave more offspring (Darwin 1871). One process by which male territorial insects compete is by direct flying contests. In butterflies, prediction of male male territorial competition for access to females has been usually based on asymmetries, for example body size (Rosenberg and Enquist 1991), body temperature (Stutt and Willmer 1998), flight morphology (Kemp 2002; Kemp et al. 2006), and prior residency (Takeuchi 2006). These factors may not directly affect the probability of winning, however, because they may not correlate with opponents physiological condition, which aids fight performance and duration, because they may change with age. One example is fat reserves in studies of contest competition in dragonflies (Marden and Waage 1990; Plaistow and Siva-Jothy 1996), in which fatter males, usually middle-aged individuals (Plaistow and Siva-Jothy 1996), win contests. Above this age fat tends to decline and is no longer a predictor of contest success. Only two studies have included assessment of energy reserves in butterflies. In Hypolimnas bolina fat reserves were not higher in old more successful males (which were assumed to have larger energy stores) during contests than in younger butterflies (Kemp 2002). Takeuchi (2006) investigated the lipid content of resident and intruder males of Chrysozephyrus smaragdinus but found no difference. Despite these studies, however, the role of fat reserves has been little explored, so whether this variable is important during butterfly territorial competition is still an open question.

2 196 J Ethol (2007) 25: The butterfly genus Eumaeus is specific to cycad plants, on which the females lay eggs and which the larval stages use as a food source (Schneider et al. 2002). Very little is known about the reproductive behavior of members of this genus. In particular, males of the butterfly Eumaeus toxea behave aggressively toward conspecific males at twilight during the reproductive season (June August) in Southeast Mexico (Solares Leal and Romo Beltrán 2001). Males often perch in higher places than cycads, for example trees and shrubs, where they encounter incoming females while also aggressively chasing conspecific males (Solares Leal and Romo Beltrán 2001). In this study, we had two objectives: 1. to describe the adult male reproductive behavior of this animal, and 2. to investigate whether size and body fat can be used to predict territorial status (resident and intruder) and/or contest success. Materials and methods The study was conducted in June 2002 and June 2003 in an outdoor site (25 m 40 m) containing approximately 200 Zamia furfuracea (Cicadaceae) plants, on which female E. toxea lay eggs (Solares Leal and Romo Beltrán 2001). The site is located in Ciénega del Sur, Alvarado, Veracruz, Mexico ( W and N). Every day, unmarked males were captured and assigned a unique number, on the underside of the right anterior wing, by use of white enamel paint. Before release, male size (length of right anterior wing from its insertion basis to the distal point) was measured using a digital calliper (Mitutoyo; precision 0.01 mm). Focal observations (1 h per individual) were conducted both to describe male contest behavior and to investigate the role of size and fat in contests. For contest behavior, observations were mainly conducted from 1600 to 2000 hours using 42 randomly chosen individuals. Special attention was paid to encounters against conspecific male individuals, and the form and duration of contests. A second set of observations was of contests that lasted longer than 17 s (long contests, see Results section), for which it was predicted that resident males (individuals that remained in the same spot for more than 1 day) and fight winners (individuals that remained in the same place after a long contest with another conspecific male) would both have a larger body size than intruders (individuals that flew briefly near a resident male) and fight losers (individuals that did not return to the cycad place after a confrontation of more than 17 s with another conspecific male). One-way ANOVA was used to test for differences between the body sizes of these groups of males. Further differences were tested by use of the Tukey test. A subsample of males of these four male categories was taken from this second set of observations to compare muscular fat. Fat was extracted from the thorax and abdomen, given that the fat content of the rest of the body (legs, wings, and head) is of little significance in insects (Anholt et al. 1991). The extraction process was based on the technique described by Plaistow and Siva-Jothy (1996) and begins with recording of the dry weight of the individual after removal of the wings, legs and head. The individual is then placed in chloroform, transferred to a dryer for evaporation of chloroform, and weighed again. The difference between the two weights is the amount of fat extracted. In the same way as for body size it was predicted that residents and winners would have more body fat than intruders and losers. To perform these comparisons a generalized linear model (GLM) was constructed. In this model, body fat was the response variable, male status (resident, intruder, winner, loser) was a factor, and body size was used as a covariate to control for allometric effects. The interaction term status size was tested but was not significant (P = 0.707). Other differences between groups were investigated by use of the least significant difference (LSD) a posteriori test. For convenience, duration of events is given in seconds and minutes. Size and fat data are given in millimeters and grams, respectively. Results are given as mean ± SD except when indicated otherwise. Statistical analysis was performed and figures were produced by use of SPSS ver. 14. Animals are deposited in the Laboratorio de Ecología de la Conducta de Artrópodos of the Instituto de Ecología (Universidad Nacional Autónoma de México). Results Males perched in trees (Prunus domesticus) above and surrounding Z. furfuracea cycads. Of 42 males observed, 30 returned to the same site and stayed there for several days (range 2 8 days). These resident individuals chased conspecific males ( intruders ) that approached within a short distance (ca. 0.5 m). In terms of duration and their non-normal distribution (Shapiro Wilk test = 0.86; P = 0.002) two types of chasing flight can be distinguished short (4.8 ± 0.9 s; N = 12) and long (17.8 ± 3.4 s; N = 13; Fig. 1). The behavioral nature of both flights was also different. In short flights the resident male chased the incoming male with the latter leaving the place without facing the resident. These encounters invariably ended with the resident male returning to its perching site (N = 12). Long fights, in contrast, took the form of spiral,

3 J Ethol (2007) 25: ascending flights with both males frequently flying several meters from the cycad. These encounters usually ended with one male, usually the resident (12 out of 13 cases), returning to the original spot. When a female passed by the male flew directly to her and grasped her with no obvious precopulatory courtship behavior. Most such encounters (27 out of 43) did not end in copulation, with females leaving the place and flying away from the site. When copulation occurred it lasted several hours (420 ± 120 min, N = 38). After copulation, males returned to their original perching spot on the trees. In two instances only females perched on the cycads and laid eggs. Males that copulated were larger than males not seen copulating (t 1,93 = 3.7; P = ; Fig. 2). All these males were residents. Given the behavioral nature and duration of the long flights we observed, we used these as true contests for winner loser analysis, as suggested by other butterfly studies (Kemp and Wiklund 2001). By use of the second set of observations (and animal collection) size differences among residents, intruders, winners, and losers were revealed (one-way ANOVA: F 3,78 = 27.51; P = ; Fig. 3), with residents and winners being larger than intruders and losers, respectively (Tukey test: P = in all cases). Resident males were not larger than winners (Tukey test: P = 0.951); similarly, intruder males were not larger than loser males (Tukey test: P = 0.067). Although fat differed significantly among males (GLM: F 3,77 = 10.19; P = ; Fig. 4) there was no significant difference between resident and intruder males (LSD: P = 0.304) or between winner and loser males (LSD: P = 0.784). The differences were because resident males had more fat than winner and loser males (LSD: P = ; P = 0.001, respectively) and intruder males were fatter than winner and loser males (LSD: P = 0.006; P = 0.001, respectively). Although fat usually increased with body size (GLM: F 1,77 = 7.81; P = 0.007; Fig. 4), closer inspection of the data revealed fat was related to body size in resident and intruder males (P = in both) but not in winner and loser males (P > in both). Discussion Fig. 1 Frequency distribution of contests according to duration E. toxea males defend spots against other males. The place they defend, as occurs with some other butterflies (e.g. Cordero and Soberón 1990), does not seem to function as a resource for females, because females were not defended against other males after copulation nor did the cycad Fig. 2 Difference (mean ± SE) between sizes of males that were observed to copulate and those never observed to copulate Fig. 3 Size differences (mean ± SE) among resident, intruder, winner, and loser males

4 198 J Ethol (2007) 25: Fig. 4 Fat (g 10 3 ) in relation to body size for resident, intruder, winner, and loser males plants seem to be guarded and exchanged for copulation. The perching spot seems to be a place where encountering females and mating is more likely, with large males being able to defend it. Our results are similar to those found for other insects males with large body size are more successful in territorial confrontations (e.g. Otronen 1988; Moore 1990). Although resident males tend to win contests, on several occasions residents are evicted. We expected conflict winners and residents to win more contests because of differences between fat reserves, because this would enable them to successfully meet the energy-consumption requirements of a fight (Marden 2000). Although fat has been correlated with contest success for some insects (e.g. Marden and Waage 1990; Plaistow and Siva-Jothy 1996), no differences between fat reserves was found for residents and intruders or for winners and losers. Residents and intruders had more fat than both winner and loser males, however. Perhaps these two last categories of male were already exhausted of energy, because fat was measured after animals had engaged in contests. The lack of a difference between the fat of residents and intruders indicates fat may not be an indicator of fighting ability. Despite this, these males did, nevertheless, have less fat than winner and loser males, which suggests fat is important in, or is affected by, the fight. These results are slightly indicative of resource storage; individuals able to sustain a flight, as a result of their better condition, will be more successful during competition. This energy perspective is surprising in butterflies, because many studies have found no support for the idea that a large size advantage results in residency advantage during contests (Kemp 2000; Takeuchi 2006). As far as we are aware this is only the third study in which fat reserves have been measured, despite a previous suggestion (Kemp and Wiklund 2001) that this variable should be investigated. Two previous studies did not find that energy stores were related to contest outcome (Kemp 2002) or residency intruder status (Takeuchi 2006). We propose that fat reserves may still be a potentially important physiological reason for the size advantage in E. toxea, given our results, in which there was a positive correlation between these two variables for resident and intruder males. Why this correlation was not present in winner and loser males is difficult to explain; possibly, and as indicated above, these males were energetically more exhausted. For this reason it is hard to assert whether fat is actually communicated between males. Fat was different after the last fight as judged by comparing values for winners and losers against those for residents and intruders, respectively (both Tukey tests, P < 0.05). These results may suggest that contests are energetically costly for opponents, and males, therefore, should be selected to perceive their competitor s energy condition. An alternative hypothesis, however, is that these energy differences may not be because of the cost of the last fight itself but because of the energy result accumulated from previous fights. In a case like this, males could perceive the energy status of their opponents. If this is so, and given the correlation between size and fat reserves, one would then expect that fight duration should be inversely correlated with the size of the resident male (so intruder males fight less if the resident is too large, i.e. has large fat reserves). Study of contest duration and the fat reserves of the 22 males that won their contest revealed a negative relationship between the two variables (r pearson = 0.46, P = 0.03). This suggests that size may be assessed by competitors and, hence, may determine contest duration, presumably because of the decision of the intruder not to engage in a long, unnecessary fight. This, of course, requires testing experimentally. In other insects in which fat has been investigated (e.g. odonates; Marden and Waage 1990; Plaistow and Siva- Jothy 1996) amounts of fat decrease with age; it is not known if this is true for E. toxea and other butterflies. Kemp and Alcock (2003) have suggested that fat reserves cannot always be used to predict the outcome of contests between insects, particularly if the animal uses a capital breeding life-history strategy relying mainly on resources gathered at the larval stage with no substantial re-fueling of energy resources at the adult stage. For Heliconius (and, possibly, H. bolina) butterflies, at least, this seems to be true, because adults re-establish their energy reserves while foraging (Karlsson 1994). Interestingly, results from work currently in progress indicate that fat reserves and body size in E. toxea adults are largely determined during larval

5 J Ethol (2007) 25: competition, so resource dependency is strong at this stage (A. Córdoba-Aguilar, unpublished data). For these reasons, fat reserves in butterfly contests would be worth studying, especially given the difficulty of finding condition-reflecting aspects of these animals (Kemp et al. 2006). Contradictory results from recent research on butterfly energetics (Kemp 2002; Takeuchi 2006), however, should not discourage further research on fat reserves in other butterflies. The only two physiological aspects that have been studied are temperature (Stutt and Willmer 1998) and flight morphology (as an indicator of flight performance ability); these are unlikely to be useful for predicting contest success (Kemp and Wiklund 2004). As long as fat declines with age in the adult, and the male does not substantially re-establish energy resources (Kemp and Alcock 2003), fat may be still a promising avenue for research in these animals. Acknowledgments To one anonymous reviewer and K. Ueda for their splendid suggestions, to Iván Sánchez-Barrera for his help in the field, and to Allari Romo Beltrán and Iliana Solares Leal for sharing some data on male fighting duration. References Andersson M (1994) Sexual selection. Princeton University Press, Princeton Anholt BR, Marden JH, Jenkins DM (1991) Patterns of mass gain in adult odonates. Can J Zool 69: Cordero C, Soberón J (1990) Non-resource based territoriality in males of the butterfly Xamia xamia (Lepidoptera: Lycaenidae). J Insect Behav 3: Darwin C (1871) The descent of man, and selection in relation to sex. Murray, London Karlsson B (1994) Feeding habits and change of body composition with age in three nymphalid butterfly species. Oikos 69: Kemp DJ (2000) Contest behavior in the speckled wood butterfly: does size matter? Behav Ecol 11: Kemp DJ (2002) Butterfly contests and flight physiology: why do older males fight harder? Behav Ecol 13: Kemp DJ, Wiklund C (2001) Fighting without weaponry: a review of male male contest competition in butterflies. Behav Ecol Sociobiol 49: Kemp DJ, Alcock J (2003) Lifetime resource utilization, flight physiology, and the evolution of contest competition in territorial insects. Am Nat 162: Kemp DJ, Wiklund C (2004) Residency effects in animal contests. Proc R Soc Lond ser B 271: Kemp DJ, Wiklund C, Van Dyck H (2006) Contest behaviour in the speckled wood butterfly (Pararge argeria): seasonal phenotypic plasticity and the functional significance of flight performance. Behav Ecol Sociobiol 59: Marden JH (2000) Variability in the size, composition and function of insect flight muscles. Ann Rev Physiol 62: Marden JH, Waage JK (1990) Escalated damselfly territorial contests are energetic wars of attrition. Anim Behav 39: Moore AJ (1990) The evolution of sexual dimorphism by sexual selection: the separate effects of intrasexual selection and intersexual selection. Evolution 44: Otronen M (1988) The effect of body size on the outcome of fights in burying beetles (Nicrophorus). Ann Zool Fenn 25: Plaistow JS, Siva-Jothy MT (1996) Energetic constrains and male mate-securing tactics in the damselfly Calopteryx splendens xanthostoma (Charpenter). Proc R Soc Lond Ser B 263:3 9 Rosenberg RH, Enquist M (1991) Contest behavior in Weidermeyer s admiral butterfly Limenitis weidemeyerii (Nymphalidae): the effects of size and residency. Anim Behav 42: Schneider D, Wink M, Sporer F, Lounibos P (2002) Cycads: their evolution, toxins, herbivores and insect pollinators. Naturwissenschaften 89: Solares Leal I, Romo Beltrán AN (2001) Ciclo biológico de Eumaeus toxea Godart. (Lepidoptera: Lycaenidae) sobre Zamia furfuracea L. (Zamiaceae). Bachelor thesis. Universidad de las Américas- Puebla, Mexico Stutt AD, Willmer P (1998) Territorial defence in speckled wood butterflies: do the hottest males always win? Anim Behav 55: Takeuchi T (2006) The effect of morphology and physiology on butterfly territoriality. Behaviour 143: